Recent intense interest in the use of rapid genetic analysis as a tool for understanding biological processes (Wodicka et al., J. Nat. Biotechnol. 15:1359-1367 (1997); Iyer et al., Science 283:83-87 (1999)), in unlocking the underlying molecular causes of disease, and in the development of biosensors, has led to a need for new sensitive and arrayable chip-based analytical tools. Of high importance is the need for techniques that do not require labeling of the target sample (Sando et al., J. Am. Chem. Soc. 124:2096-2097 (2002), since that increases the time, cost, and potential for error inherent in the analysis. In the context of solution-phase assays, the molecular beacon concept has proven itself to be both sensitive and reliable (Broude, Trends Biotech. 20:249-256 (2002); Dubertret et al., Nature Biotech. 19:365-370 (2001)). Molecular beacons consist of a DNA hairpin functionalized at one end with a fluorophore, and at the other with a quenching agent (Tyagi et al., Nat. Biotechnol. 14:303-308 (1996); Joshi et al., Chem. Commun. 1(6):549-550 (2001)). In the absence of the target DNA sequence, the quencher is brought in close proximity to the fluorophore, and no signal is generated. Addition of the target sequence leads to hairpin unfolding, concomitant duplex formation, and signal generation.
Although a few reports of surface-immobilized molecular beacons have appeared in the literature (Fang et al., J. Am. Chem. Soc. 121:2921-2922 (1999); Wang et al., Nucl. Acids. Res. 30:e61 (2002)), it is believed that these approaches employ an attached single molecule as quencher, while the material on (or in) which the hairpin is immobilized serves only a passive role. As part of a general program aimed at developing “label-free” optical biosensors (Chan et al., J. Am. Chem. Soc. 123:11797-11798 (2001)), it was of interest, therefore, to investigate whether the substrate material itself could be used as a quenching agent. Using the substrate itself as the quenching agent significantly decreases the complexity of the synthetic DNA probe hairpin, because attachment of a separate quencher is unnecessary. This approach has been successfully demonstrated using planar gold surfaces that effectively serve as both anchoring points and quenchers for DNA hairpin probes bearing an attached fluorophore (see U.S. Patent Publ. No. 20070059693 and co-pending U.S. patent application Ser. No. 11/838,616).
One straightforward approach for improving the sensitivity of the DNA hairpin array is to increase the intensity of the fluorescence for a given excitation intensity. Simply increasing the intensity of the excitation source is not a viable route to increased signals, because many fluorescent labels will photobleach completely within a fraction of a second for high excitation powers. For example, because rhodamine—comparatively a very photostable dye—photobleached at I˜1 kW/cm2, it is highly unlikely that changing dyes will improve the signal. Clearly, new approaches are needed to increase the signal from these devices.
An additional limitation arises when using metal films as quenching surfaces for fluorophore-functionalized DNA hairpin probes. Typically, these films are sufficiently thick that light does not pass through them (i.e., they are opaque or near-opaque). This constrains visualization of the chip to only one side. It would be desirable, therefore, to prepare a sensor chip that is not so constrained.
The present invention is directed to overcoming these and other deficiencies in the art.